International Journal of Systematic and Evolutionary Microbiology (2002), 52, 2219–2230
DOI : 10.1099/ijs.0.01408-0
Characterization of rhizobia that nodulate
legume species of the genus Lespedeza and
description of Bradyrhizobium yuanmingense
sp. nov.
1
College of Biological
Sciences, China
Agricultural University,
Beijing 100094, People’s
Republic of China
2
Departamento de
Microbiologı! a, Escuela
Nacional de Ciencias
Biologicas, Instituto
Polite! cnico Nacional, Prol.
de Carpio y Plan de Ayala,
11340 Me! xico D. F., Mexico
3
Department of Resource
and Environment,
Northwest Science and
Technology University of
Forestry and Agriculture,
Yangling, People’s
Republic of China
Zhu Yun Yao,1 Feng Ling Kan,1 En Tao Wang,2 Ge Hong Wei1,3
and Wen Xin Chen1
Author for correspondence : Wen Xin Chen. Tel : j86 10 6289 1854. Fax : j86 10 6289 1055.
e-mail : wenxinIchen!263.net
Legume species belonging to the genus Lespedeza are annual or perennial herb
or shrub plants that grow in the northern hemisphere. They are known for the
formation of root nodules, but little information is available about their
microsymbionts. In this study, 58 root-nodule isolates from Lespedeza spp.,
obtained from China and the USA, were characterized using numerical
taxonomic analysis of phenotypic features, SDS-PAGE analysis of whole-cell
proteins, DNA–DNA hybridization, 16S rRNA gene sequence analysis and crossnodulation with selected legume species. From the results generated using
these approaches, it was concluded that Lespedeza spp. were promiscuous
hosts for rhizobia. Four main clusters of bacteria, which included 35 of the
strains isolated from Lespedeza spp., were defined upon numerical taxonomic
analysis ; these groups corresponded to those determined from analyses of
protein electrophoretic and DNA–DNA hybridization data. The four clusters
were found to define strains belonging to one of four species, Sinorhizobium
saheli, Bradyrhizobium japonicum, Bradyrhizobium elkanii or a novel species of
the genus Bradyrhizobium. The strains of B. japonicum and B. elkanii were all
from the USA soil samples, and their representative strains could not nodulate
soybean. The seven strains found to represent the novel Bradyrhizobium sp.
were from China. These were differentiated from recognized species of the
genus Bradyrhizobium by all of the taxonomic methods used here ; hence, it is
proposed that the novel strains isolated from Lespedeza spp. represent a novel
species of the genus Bradyrhizobium, Bradyrhizobium yuanmingense. The type
strain of the novel species, CCBAU 10071T (l CFNEB 101T), formed ineffective
nodules on Medicago sativa and Melilotus albus but did not nodulate soybean.
The other 23 bacterial strains isolated from Lespedeza spp. were found to form
single branches or small groups (two to three strains) that were related to
Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium spp. on the
basis of numerical taxonomic analysis, indicating the possibility that other
rhizobial species are also associated with Lespedeza spp.
Keywords : Lespedeza, Bradyrhizobium, Sinorhizobium, diversity, phylogeny
INTRODUCTION
Legume species of the genus Lespedeza are annual or
perennial wild plants, of which there are known to be
.................................................................................................................................................
The GenBank accession number for the 16S rRNA gene sequence of
Bradyrhizobium yuanmingense CCBAU 10071T is AF193818.
around 140 species of herbs or shrubs. Most of these
species (125) are native to eastern Asia and about 15
are indigenous to the south-eastern United States
(Allen & Allen, 1981). Most Lespedeza spp. are
drought-enduring plants, and are held in high esteem
as foliage, green manure crops or honey resources and
in preventing soil erosion (Allen & Allen, 1981). On
01408 # 2002 IUMS Printed in Great Britain
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Z. Y. Yao and others
some de-forested hills in north-eastern China, Lespedeza spp. are the main pioneer plants after the trees
have been cut ; hence, they may be potential plants for
re-forestation. Different Lespedeza spp. are used in
Chinese herbal medicine, e.g. Lespedeza cyrtobotrya,
Lespedeza buergeri, Lespedeza davidii, Lespedeza
bicolor and Lespedeza cuneata are used for treating
coughs and fevers, and Lespedeza chinensis is used for
treating diarrhoea (He, 1993). Clinical tests using
constituents (flavonoids or extracts) from Lespedeza
spp. have been performed (Campanacci et al., 1965).
Research has also been done on the biological degradation of tannins in sericea lespedeza (L. cuneata) by
white rot fungi, in an attempt to increase the potential
value of L. cuneata as a forage crop (Gamble et al.,
1996). Previous studies on cross-nodulation (performed between 1917 and 1974 ; summarized by Allen
& Allen, 1981) indicated that Lespedeza plants belonged to the cowpea miscellany. Marked host specificity relative to the effectiveness of nodulation was
also revealed in these previous studies. The perennial
Lespedeza spp. showed effective responses to bacterial
strains isolated from perennial species, while the
annual species had effective responses to the strains
isolated from annual species (Allen & Allen, 1981).
The microsymbionts of the leguminous plants of the
cowpea miscellany were classified as belonging to the
bacterial genus Bradyrhizobium, by using a taxonomic
approach (Jordan, 1984). However, the specific taxonomic positions for most of the bacteria associated
with the cowpea miscellany were unknown at that
time. Since its creation in 1982 (Jordan, 1982), three
species have been assigned to the genus Bradyrhizobium : Bradyrhizobium japonicum (Jordan, 1982), Bradyrhizobium elkanii (Kuykendall et al., 1992) and
Bradyrhizobium liaoningense (Xu et al., 1995).
So far, about 70 species of Lespedeza have been
recorded in China (He, 1993). However, the nodulation of these species and their microsymbionts have
not been documented. During a survey of rhizobial
resources in the northern parts of China, we obtained
a number of rhizobial isolates from a variety of
different plants and geographical regions. The characterization of some populations of these isolates according to their geographical origin or host has been
done already (Chen et al., 1995 ; Tan et al., 1999 ; Wang
et al., 1999). Because of the potential values of
Lespedeza spp. in agriculture and re-forestation and
the uncertain taxonomic position of the rhizobial
species associated with them, we decided to characterize the rhizobial strains isolated from these plants
using a polyphasic approach. The aims of this research
were to check the diversity of these strains and to
classify them.
strains were used : B. liaoningense FSI 2062T (Xu et al.,
1995) ; B. japonicum USDA 6T (Jordan, 1982) ; B. japonicum
USDA 110 (Gao et al., 1994) ; B. japonicum B15 (Gao et al.,
1994) ; B. elkanii USDA 76T (Kuykendall et al., 1992) ;
Mesorhizobium loti NZP 2213T (Jarvis et al., 1997) ; Mesorhizobium huakuii CCBAU 2609T (Chen et al., 1991) ;
Mesorhizobium ciceri UPM-Ca7T (Nour et al., 1994) ; Mesorhizobium tianshanense A-1BST (Chen et al., 1995) ; Mesorhizobium amorphae ACCC 19665T (Wang et al., 1999) ;
Mesorhizobium plurifarium USDA 4413 (de Lajudie et al.,
1998b) ; Rhizobium leguminosarum USDA 2370T (Jordan,
1984) ; Rhizobium tropici type B CIAT 899T (Martı! nezRomero et al., 1991) ; Rhizobium tropici type A CFN 299
(Martı! nez-Romero et al., 1991) ; Rhizobium etli CFN 42T
(Segovia et al., 1993) ; Rhizobium galegae HAMBI 540T
(Lindstro$ m, 1989) ; Sinorhizobium meliloti USDA 1002T
(Jordan, 1984) ; Sinorhizobium fredii USDA 205T (Scholla &
Elkan, 1984) ; Sinorhizobium saheli USDA 4102T (de Lajudie
et al., 1994) ; Sinorhizobium terangae USDA 4101T (l ORS
1037T) (de Lajudie et al., 1994) ; Escherichia coli DH5α
(Sambrook et al., 1989). Plasmid pUC18 (Sambrook et al.,
1989) was also used as a vector for cloning. The Chinese
strains were isolated in six provinces or cities in northern
China, all within an area of about 2000 km east to west by
1000 km north to south. The provinces of Gansu, Shanxi
and Shaanxi are located in the Loess Plateau in northwestern China ; they have a semi-arid climate and soils that
are poor in organic matter. The climate and soils of Beijing
and Inner Mongolia are similar to those of Shanxi, but these
regions have more rain in the summer. Jilin is a province in
north-eastern China ; the soils in this region contain more
organic matter and water, but the region has very cold
winters (below k20 mC). The strains isolated in the USA
were obtained from the USDA-ARS Rhizobium Germplasm Resource Collection (United States Department of
Agriculture) ; some of these strains were isolated over half a
century ago (Table 1). The symbiotic ability of each novel
strain was confirmed by nodulating its original host using
the method of Vincent (1970). Of the 15 host species tested,
Lespedeza stipulacea and Lespedeza striata are annual herbs ;
the others are perennial plants.
Phenotypic
characterization
and numerical
taxonomy.
METHODS
Phenotypic features, i.e. the utilization of sole carbon sources
and sole nitrogen sources, resistance to antibiotics, tolerance
to dyes and chemicals, tolerance to NaCl, temperature and
pH ranges for growth, and some physiological and biochemical reactions, were determined using protocols described previously (Gao et al., 1994). The results of the
phenotypic characterization were converted into a binary
dataset which was used to estimate the simple matching
similarity coefficient (Ssm) of each strain pair and to generate
a similarity matrix (Sneath & Sokal, 1973). The similarity
matrix was used for cluster analysis to construct a dendrogram using the unweighted pair group method with averages
(UPGMA) (Sneath & Sokal, 1973). The mean similarity for
each strain within a cluster was estimated to present the
phenotypic variation in the cluster, as described previously,
and the central strain as the representative for the phenotypic
cluster was calculated (Sneath & Sokal, 1973). Generation
times were determined in PY medium (5 g peptone of casein,
3 g yeast extract, 0n6 g CaCl ; all vales given are per litre of
distilled water) by using #a spectrophotometric method
(Vincent, 1970).
Isolates and strains. Fifty-eight nodule strains isolated from
SDS-PAGE of whole-cell proteins. Soluble-protein extraction,
15 Lespedeza spp. that grow in the USA and in Chinese soils
were used in this study (Table 1). The following reference
SDS-PAGE and scanning of the protein patterns using a
Densitometer Extra-Scanner (LKB) were done as described
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Bradyrhizobium yuanmingense sp. nov.
Table 1. Bacterial strains used in this study
Lespedeza host species
Strain
Cluster 1 (Sinorhizobium saheli)
CCBAU 10012, CCBAU 10032, CCBAU 10043, CCBAU 10063,
CCBAU 10171, CCBAU 10091, CCBAU 10113, CCBAU 10143,
CCBAU 10152, CCBAU 10155, CCBAU 10163
CCBAU 13072, CCBAU 13074
CCBAU 13145
CCBAU 13146
Cluster 2 (Bradyrhizobium yuanmingense)
CCBAU 10011, CCBAU 10030, CCBAU 10031, CCBAU 10038,
CCBAU 10040, CCBAU 10071T, CCBAU 10073
Cluster 3 (Bradyrhizobium japonicum)
USDA 3399
USDA 3638
USDA 3651
USDA 3652
USDA 3654
Cluster 4 (Bradyrhizobium elkanii)
USDA 3199
USDA 3203
USDA 3204
USDA 3205
USDA 3211
USDA 3212
USDA 3220
USDA 3222
Other Bradyrhizobium strains
CCBAU 10033
USDA 3198
USDA 3202
USDA 3208
USDA 3219
Other fast or moderately growing rhizobial strains
USDA 3197
USDA 3215
CCBAU 10201, CCBAU 10241, CCBAU 10301
CCBAU 73041
CCBAU 13008, CCBAU 13132
CCBAU 13143
CCBAU 10112, CCBAU 10134
CCBAU 01104
CCBAU 71055
CCBAU 71082
CCBAU 03208, CCBAU 03216
CCBAU 03220, CCBAU 03248
previously (Tan et al., 1997). Clustering analysis of the
protein patterns was performed as described previously (Tan
et al., 1997).
Analyses of 16S rRNA gene sequence data and phylogeny.
These were performed only for the representative strain of
the novel species, strain CCBAU 10071T (Cluster 2). The
complete 16S rRNA gene (" 1500 bp) was amplified by
PCR using the universal forward primer P1 (5h-CGggatcc-
Geographical origin
(year of isolation)
L. inschanica
Beijing, China (1995)
L. daurica
L. tomentosa
L. cyrtobotrya
Jilin, China (1995)
Jilin, China (1995)
Jilin, China (1995)
L. cuneata
Beijing, China (1995)
L. juncea var. sericea
L. juncea
L. daurica
L. daurica
L. juncea
USA (unknown)
OH, USA (1960)
USA (1960)
MO, USA (1960)
USA (1960)
L. striata
L. capitata
L. capitata
L. stipulacea
L. juncea var. sericea
L. juncea var. sericea
L. procumbens
L. bicolor
MD, USA (1933)
OH, USA (1940)
USA (1932)
NJ, USA (1933)
VA, USA (1914)
TN, USA (1933)
SC, USA (1941)
SC, USA (1940)
L. cuneata
L. striata
L. capitata
L. stipulacea
L. juncea var. serpens
Beijing, China (1995)
USA (unknown)
OH, USA (1940)
IN, USA (1938)
SC, USA (1941)
L. hirta
L. striata
L. daurica
L. formosa
L. bicolor
L. hydysaroides
L. inschanica
L. bicolor
L. daurica
L. bicolor
L. floribunda
L. daurica
OH, USA (1940)
MD, USA (1937)
Beijing, China (1995)
Gansu, China (1995)
Jilin, China (1995)
Jilin, China (1995)
Beijing, China (1995)
Inner Mongolia, China (1995)
Shaanxi, China (1995)
Shaanxi, China (1995)
Shanxi, China (1995)
Shanxi, China (1995)
AGFGTTTGATCCTGGTCAGAACGAACGCT-3h) and
the universal reverse primer P6 (5h-CGggatccTACGGCTACCTTGTTACGACTTCACCCC-3h) and a standard protocol. The PCR product was cloned into pUC18, which was
then transformed into E. coli DH5α (Sambrook et al., 1989),
as described by Tan et al. (1997). An automated DNA
sequencer and primers P1, P2, P3, P4, P5 and P6 (Tan et al.,
1997) were used for single-strand sequencing of the cloned
16S rRNA gene fragments, after purification of the plasmids.
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The 16S rRNA gene sequence of CCBAU 10071T and the
16S rRNA gene sequences of its related bacterial species
(retrieved from GenBank) were aligned by using the program
 in the GCG package (Genetics Computer Group,
1995). Sequence similarities and a phylogenetic tree were
obtained using   (Thompson et al., 1994). The
phylogenetic tree constructed was visualized by using 
 (Page, 1996).
Determination of DNA GjC content and DNA–DNA homology. DNA was prepared by a standard phenol\chloroform
extraction (Marmur, 1961). The DNA GjC content of
strains was determined using the thermal melting protocol of
De Ley (1970), using E. coli K-12 DNA as the standard.
DNA–DNA homologies were estimated spectrophotometrically (De Ley et al., 1970). DNA was sheared by passage
through a syringe, to generate fragments of 2–5i10& Da in
size. Renaturation was performed in 2iSSC at the optimum
temperature (De Ley et al., 1970).
PCR-based RFLP analysis of 16S rDNA. Primers fD1 and rD1
(Weisburg et al., 1991) and procedures described previously
(Wang et al., 1999) were used for amplification of the
complete 16S rRNA genes, for digestion of the PCR
products with MspI, HinfI, HhaI or Sau3AI, and for
separation of the digested fragments in 3n0 % (w\v) agarose
gels.
Cellular plasmid contents. The Eckhardt (1978) procedure
modified by Hynes & McGregor (1990) was used to visualize
any plasmids present in the strains.
Cross-inoculation tests. Standard protocols were used (Vincent, 1970). Plants were grown in pots filled with vermiculite
moistened with a nitrogen-free plant nutrition solution
(Vincent, 1970). Nodulation and nitrogen-fixing abilities of
the plants were observed after 1 month. The recommended
legume species of Graham et al. (1991), Medicago sativa,
Melilotus albus, Vigna unguiculata, Glycyrrhiza uralensis,
Glycine max, Phaseolus vulgaris, Pisum sativum, Galega
officinalis, Trifolium repens and Leucaena leucocephala, were
used as hosts for strain CCBAU 10071T, which was isolated
from Lespedeza cuneata and was the representative strain of
the novel Bradyrhizobium sp. described here. L. cuneata was
inoculated with S. saheli USDA 4102T, B. japonicum USDA
6T and B. elkanii USDA 76T. Glycine max was inoculated
with USDA 3204 and USDA 3654, representatives of the B.
japonicum and B. elkanii strains, respectively, isolated from
Lespedeza spp.
RESULTS
Isolation
The growth rate of and acid\alkali production by each
strain were determined on YMA medium (Vincent,
1970) plates during the isolation procedure. Of the 58
strains isolated, 30 from China and two from the USA
were fast-growing, acid-producing rhizobia that produced colonies 2 mm in diameter after 3–5 days
incubation. Eight strains from China and 17 from the
USA were slow-growing, alkali-producing bacteria
that belonged to the genus Bradyrhizobium, with
colonies 1 mm in diameter after 7–10 days incubation (Table 1).
2222
Phenotypic characterization and numerical taxonomy
For all of the strains (including the reference strains),
125 phenotypic features were tested. None of the
strains tested used malonate, oxalate, sorbose or methionine as a sole carbon source, and none used alanine, -glutamine or -threonine as a sole nitrogen
source. All of the strains were resistant to 5 µg
ampicillin ml−", 5 µg chloramphenicol ml−", 5 µg erythromycin ml−" and 0n1 % Congo red ; they were
sensitive to 100 µg kanamycin ml−", 300 µg erythromycin ml−", 300 µg neomycin ml−" and 0n1 % crystal
violet and methylene blue. They were all sensitive to
heat treatment at 60 mC for 10 min, and were catalasepositive. The 105 features found to be variable among
the strains were used for the numerical taxonomic
analysis. Based on the results of cluster analysis (Fig.
1), the levels of similarity between most of the type
strains of recognized species were less than 80 %. The
exceptions were Mesorhizobium ciceri and Mesorhizobium huakuii, which were separated at the 83 %
similarity level. Thirty-five of the 58 strains isolated
from Lespedeza spp. belonged to one of four clusters,
with around 80 % similarity ; other strains formed
single branches or small groups (of two to three strains)
that consisted of the strains isolated from Lespedeza
spp. only or which were related to recognized species
(Fig. 1). Some important features of the four clusters
are described below.
Cluster 1 consisted of 15 fast-growing strains isolated
from four species of Lespedeza (L. inschanica, L.
daurica, L. tomentosa and L. cyrtobotrya) that grow in
the Beijing and Jilin Provinces of China ; S. saheli ORS
609T was also found in this cluster. The mean similarities for the strains ranged from 82n0 %, in the case
of strain CCBAU 13145, to 88n2 %, in the case of strain
CCBAU 10091 – this latter strain was chosen as the
central strain for the cluster. Cluster 1 strains produced
colonies that were 2 mm in diameter after 2–3 days
incubation at 28 mC. All of the strains in this cluster
produced acid when grown on YMA plates. They were
resistant to 100 µg erythromycin ml−", but sensitive to
50 µg kanamycin ml−", 300 µg erythromycin ml−" and
300 µg polymyxin ml−". Growth of Cluster 1 strains
was obtained on YMA supplemented with 0n1 %
Congo red, deoxycholate or nitrite, but not with crystal
violet or methylene blue. The strains grew on YMA
supplemented with 1 % (w\v) NaCl and at pH 5–10.
No nitrate reduction or growth in Luria–Bertani broth
was observed for Cluster 1 strains.
The seven strains of Cluster 2 were slow-growing
rhizobia that had been isolated from L. cuneata found
in Beijing. Generation times for strains CCBAU 10011,
CCBAU 10038, CCBAU 10040, CCBAU 10071T and
CCBAU 10073 were 12n5, 16n0, 9n5, 10n2 and 10n2 h,
respectively. The mean similarities for the seven strains
ranged from 83n3 to 89n2 %. CCBAU 10071T was
designated the representative strain of the cluster.
Cluster 2 strains produced single colonies of 1–2 mm in
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Bradyrhizobium yuanmingense sp. nov.
Strain
CCBAU 10012
CCBAU 13146
CCBAU 10032
CCBAU 10043
CCBAU 10091
CCBAU 10063
CCBAU 10113
CCBAU 10171T
CCBAU 10163
CCBAU 10143
CCBAU 10152
CCBAU 13072
CCBAU 10155
ORS 609T
CCBAU 13074
CCBAU 13145
CCBAU 10008
CCBAU 13132
CCBAU 10134
ORS 1009T
CCBAU 10112
USDA 205T
CCBAU 01104
USDA 2370T
USDA 1002T
CCBAU 03220
UPM-Ca7T
CCBAU 2609T
CCBAU 03248
CIAT 899T
CCBAU 10301
CCBAU 71055
CCBAU 13143
USDA 3215
USDA 3197
CCBAU 10201
CCBAU 10241
CCBAU 73041
CCBAU 71082
HAMBI 540T
CCBAU 03208
NZP 2213T
A-1BST
CCBAU 71082
CCBAU 10030
CCBAU 10011
CCBAU 10031
CCBAU 10038
CCBAU 10040
CCBAU 10071
CCBAU 10073
USDA 3399
USDA 3651
USDA 3654
USDA 3652
USDA 3638
USDA 6T
USDA 3202
USDA 3219
USDA 3203
USDA 3204
USDA 3220
USDA 3205
USDA 3211
USDA 3222
USDA 3199
USDA 3212
USDA 3208
CCBAU 10033
USDA 3198
60
65
70
75 80 85
Similarity (%)
90
95
Host
L. inschanica
L. cyrtobotrya
L. inschanica
L. inschanica
L. inschanica
L. inschanica
L. inschanica
L. inschanica
L. inschanica
L. inschanica
L. inschanica
L. daurica
L. inschanica
L. daurica
L. tomentosa
L. bicolor
L. bicolor
L. inschanica
Cluster and species
1 S. saheli
S. terangae
L. inschanica
S. fredii
L. bicolor
R. leguminosarum
S. meliloti
L. daurica
M. ciceri
M. huakuii
L. daurica
R. tropici
L. daurica
L. daurica
L. hydysaroides
L. striata
L. hirta
L. daurica
L. daurica
L. formosa
L. bicolor
R. galegae
L. floribunda
M. loti
L. floribunda
L. cuneata
L. cuneata
L. cuneata
L. cuneata
L. cuneata
L. cuneata
L. cuneata
L. juncea
L. daurica
L. juncea
L. daurica
L. juncea
L. capitata
L. juncea
L. capitata
L. capitata
L. procumbens
L. stipulacea
L. juncea
L. bicolor
L. striata
L. juncea
L. stipulacea
L. cuneata
L. striata
M. tianshanense
2 B. yuanmingense
3 B. japonicum
4 B. elkanii
100
diameter after 7–10 days incubation, and alkali when
grown on YMA or in litmus milk. They were resistant
to 300 µg polymyxin ml−", but sensitive to 100 µg
.....................................................................................................
Fig. 1. Dendrogram constructed based on
numerical taxonomic analysis using the
simple matching similarity coefficient (Ssm)
and the UPGMA (Sneath & Sokal, 1973).
Four clusters were recovered among the
strains isolated from Lespedeza spp. at the
80 % similarity level.
erythromycin ml−", 1 % NaCl (w\v) and pH 5 and 10.
Nitrate was reduced by all seven strains. None of the
Cluster 2 strains grew in Luria–Bertani broth.
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Cluster 3 contained five slow-growing strains isolated
from L. daurica and Lespedeza juncea in the USA ; B.
japonicum USDA 6T was also found in this cluster. The
strains within this cluster had mean similarities of
80n0–83n6 %. Strain USDA 3654 was designated the
representative strain of the cluster. Cluster 3 strains
produced alkali when grown on YMA, and formed
single colonies of
1 mm in diameter after 7 days
incubation on YMA plates. Similar to Cluster 2 strains,
Cluster 3 strains were resistant to 300 µg polymyxin
ml−" and sensitive to 100 µg erythromycin ml−". No
growth was observed in Luria–Bertani broth or on
YMA supplemented with 1 % (w\v) NaCl or at pH 5
and 10. All five of the strains reduced nitrate.
Eight slow-growing strains isolated from Lespedeza
bicolor, Lespedeza capitata, L. juncea var. sericea,
Lespedeza procumbens and L. striata in the USA were
grouped in Cluster 4. They had mean similarities of
80n0–84n1 %. Strain USDA 3204 was designated the
representative strain of the cluster. Cluster 4 strains
produced single colonies of 1 mm in diameter after
1 week incubation on solid medium, and produced
alkali when grown on YMA plates. They were resistant
to 100 µg erythromycin ml−" and 300 µg polymyxin
ml−", and tolerant to 1 % NaCl and pH 5. Some of the
strains could grow at pH 10. No nitrate reduction or
growth in Luria–Bertani broth was observed for the
Cluster 4 strains.
Five of the slow-growing, alkali-producing strains
isolated from Lespedeza spp. showed 67–77 % similarity with the Bradyrhizobium spp. clusters (Fig. 1),
but they could not be included in these clusters. Strains
USDA 3202, USDA 3219 and USDA 3208 had
similarities of 76–77 % with Cluster 4. Strains CCBAU
10033 and USDA 3198 formed single branches that
were divergent from each other and from Clusters 1–4.
The other 17 strains isolated from Lespedeza spp. were
fast-growing or moderately growing, acid-producing
rhizobia that had closer relationships with Mesorhizobium, Rhizobium or Sinorhizobium spp. than the other
strains isolated (Fig. 1). Strain CCBAU 10134, isolated
from L. inschanica, had 87 % similarity with S.
terangae USDA 4101T. Strains CCBAU 10112 and
CCBAU 01104 clustered with S. fredii USDA 205T at
the 83 % similarity level. Strain CCBAU 03220 shared
80 % similarity with Mesorhizobium ciceri UPM-Ca7T
and Mesorhizobium huakuii CCBAU 2609T. Strain
CCBAU 71082 was 83 % similar to R. galegae HAMBI
540T. The remaining 12 strains either formed strain
pairs or single branches, which had similarities of
71–80 % with the recognized species.
SDS-PAGE of soluble proteins
Representative strains and reference strains were
selected based on the clustering results of the numerical
taxonomic analysis. The grouping of strains based on
protein patterns (data not shown) was in general
2224
5%
Rhizobium tropici LMG 9517 (X67234)
100
Rhizobium tropici CIAT 899T (U89832)
57
Rhizobium rhizogenes IFO 13257T (D01257)
85
97
Rhizobium etli CFN 42T (U28916)
Rhizobium leguminosarum USDA 2370T (U29386)
51 Rhizobium gallicum R602spT (U86343)
99
Rhizobium yanglingense CCBAU 71623T (AF003375)
Rhizobium mongolense USDA 1844 (U89817)
Rhizobium hainanense I66T (U71078)
Rhizobium vitis NCPPB 3554T (D01258)
Rhizobium undicola LMG 11875T (Y17047)
100 Rhizobium radiobacter NCPPB 2437 (D01256)
79 80
Rhizobium rubi IFO 13261T (D14503)
62 Rhizobium sp. OK-55 (D14510)
100 Rhizobium huautlense SO2T (AF025852)
88
Rhizobium galegae ATCC 43677T (D11343)
Rhizobium giardinii H152T (U86344)
63 Sinorhizobium saheli LMG 7837T (X68390)
Sinorhizobium terangae LMG 7834T (X68388)
100
99 Sinorhizobium xinjiangense IAM 14142T (D12796)
Sinorhizobium fredii ATCC 35423T (D01272)
T
100 Sinorhizobium medicae A 321 (L39882)
Sinorhizobium meliloti LMG 6133T (X67222)
100
78 Mesorhizobium tianshanense A-1BST (AF041447)
62 Mesorhizobium mediterraneum UPM-Ca36T (L38825)
Mesorhizobium amorphae ACCC 19665T (AF041442)
T
78 Mesorhizobium huakuii IFO 15243 (D13431)
Mesorhizobium plurifarium LMG 11892T (Y14158)
99
Mesorhizobium chacoense PR5 (AJ278249)
100 Mesorhizobium loti LMG 6125T (X67229)
98
Mesorhizobium ciceri UPM-Ca7T (U07934)
100
100 Phyllobacterium mysinacearum IAM 13584 (D12789)
Phyllobacterium rubiacearum IAM 13587 (D12790)
90 Bradyrhizobium japonicum LMG 6138T (X66024)
99 Bradyrhizobium liaoningense LMG 18230T (AJ250813)
63
Bradyrhizobium yuanmingense CCBAU 10071T (AF193818)
69
Bradyrhizobium japonicum USDA 110 (D13430)
76
Rhodopseudomonas palustris ATCC 17001T (D25312)
79
Blastobacter denitrificans LMG 8443T (S46917)
84
Afipia felis (M65248)
100
Afipia clevelandensis (M69186)
Bradyrhizobium sp. SH 283012 (AF041446)
Bradyrhizobium elkanii USDA 76T (U35000)
55
Azorhizobium caulinodans LMG 6465T (X67221)
71
83
.................................................................................................................................................
Fig. 2. Dendrogram showing the phylogenetic position of
strain CCBAU 10071T (Cluster 2 ; Fig. 1) with respect to the
genus Bradyrhizobium and related genera. 16S rRNA gene
sequences were aligned using the PILEUP program in the GCG
package (Genetics Computer Group, 1995). CLUSTAL W
(Thompson et al., 1994) was used to construct the tree ;
bootstrap values are shown at the nodes of the tree, and are
expressed as a percentage of 1000 replications. Bar, 5
nucleotide substitutions per 100 nucleotides.
agreement with the groupings seen in the numerical
taxonomic analysis. S. saheli USDA 4102T, B. japonicum USDA 6T and B. elkanii USDA 76T clustered with
strains of Clusters 1, 3 and 4, respectively. The
similarities of the protein patterns ranged from 79 to
94n5 % in Cluster 1 (including S. saheli USDA 4102T),
90n5 to 94 % in Cluster 2, 78 to 90 % in Cluster 3
(including B. japonicum USDA 6T) and 80 to 93n5 % in
Cluster 4 (including B. elkanii USDA 76T). The
similarities between strains of the different clusters
were 68 %.
16S rRNA gene sequence and phylogenetic analyses
Two independent 16S rRNA gene clones from strain
CCBAU 10071T were sequenced ; the sequences from
the two clones were identical. The 16S rRNA gene
sequence from strain CCBAU 10071T had 99n2, 98n2,
96n4 and 95n2 % similarity with 16S rDNA sequences
from B. japonicum USDA 6T, Bradyrhizobium sp.
USDA 110, B. elkanii USDA 76T and Bradyrhizobium
sp. (Amorpha) SH 283012 (Wang et al., 1999), respectively. The phylogenetic relationships between the
recognized species in the phylogenetic tree we constructed (Fig. 2) were the same as those reported
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Bradyrhizobium yuanmingense sp. nov.
Table 2. DNA GjC content and DNA–DNA relatedness values for strains used in this study
.................................................................................................................................................................................................................................................................................................................
Strains : 1, S. saheli CCBAU 10091 ; 2, B. yuanmingense CCBAU 10071T ; 3, B. japonicum USDA 6T ; 4, B. elkanii USDA 3204 ;
5, B. elkanii USDA 76T. For each strain, the DNA GjC content was determined by the thermal denaturation method (Tm) ;
DNA–DNA relatedness was determined from initial reassociation rates. Data shown represent the mean values of duplicate
hybridizations performed using the spectrophotometric method of De Ley et al. (1970). , Not determined.
Strain
DNA GjC content (mol %)
DNA–DNA homology (%) with
1
Cluster 1 (S. saheli)
CCBAU 10032
CCBAU 10043
CCBAU 10063
CCBAU 10171
CCBAU 10091
CCBAU 10113
CCBAU 10143
CCBAU 10152
CCBAU 10163
CCBAU 13072
CCBAU 13074
CCBAU 13145
CCBAU 13146
ORS 609T
Cluster 2 (B. yuanmingense)
CCBAU 10011
CCBAU 10030
CCBAU 10031
CCBAU 10038
CCBAU 10040
CCBAU 10071T
CCBAU 10073
Cluster 3 (B. japonicum)
USDA 3399
USDA 3638
USDA 3651
USDA 3652
USDA 3654
USDA 6T
B15
USDA 110
Cluster 4 (B. elkanii)
USDA 3203
USDA 3204
USDA 3205
USDA 3211
USDA 3212
USDA 3220
USDA 3222
USDA 3199
USDA 76T
B. liaoningense SFI 2032T
64n1
62n4
62n2
64n1
62n4
63n1
63n0
61n8
62n0
63n9
63n7
62n2
61n8
63n3


63n8
63n9
64n5
63n9
62n8
62n9
63n9
62n1
63n9
previously (e.g. Wang et al., 1999 ; Willems & Collins,
1993 ; Young & Haukka, 1996). Species of the genera
Mesorhizobium (Jarvis et al., 1997) and Sinorhizobium
(Chen et al., 1988 ; de Lajudie et al., 1994) clustered in
2
3
72n6
75n4
72n8
81n0
90n2
100
90n6
10n0
4
5
80n8
91n0
100
100
100
100
72n2
100
76n5
100
92n5
100
89n6
89n6
29n8
23n9
45n2
25n3p16n8
17n3
47n8
46n2
42n7
32n3
15n8
11n4
88n6
87n0
72n0
71n4
79n8
100
0
94n5
100
78n0
79n6
100
86n6
72n0
80n3
91n1
two monophyletic groups that were distinct from other
genera. The remaining fast-growing, acid-producing
bacteria clustered in the genus Rhizobium (Young et
al., 2001) [formerly comprising the genera Allorhizo-
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Z. Y. Yao and others
Table 3. Characteristics useful for distinguishing B. yuanmingense from related species
.....................................................................................................................................................................................................................................
Strains : 1, B. yuanmingense (Cluster 2, n l 7) ; 2, B. elkanii (Cluster 4, n l 8) ; 3, B. japonicum
(Cluster 3, n l 6) ; 4, B. liaoningense. , Not reported ; , not done. Data from this study for
B. yuanmingense, B. elkanii and B. japonicum and from Xu et al. (1995) for B. liaoningense. k,
95 % isolates negative ; j, 95 % isolates positive ; d, 5–95 % isolates positive.
Characteristic
Carbon sources used :
-Fructose
Inositol
Lactose
Maltose\sucrose
Nitrogen sources used :
(j)-Aspartic acid
Glycine
Resistant to :
Erythromycin (100 µg ml−")
Polymyxin (300 µg ml−")
0n1 % Methyl green\deoxycholate
1n0 % NaCl
Growth at pH 5n0
Growth at pH 10n0
Nitrate reduction
Colony size after 7–10 days incubation (mm)
Generation time (h)
GjC content of DNA (mol %)
Soybean-nodulating
L. cuneata-nodulating
16S rRNA gene PCR-RFLP pattern
1
2
3
4
k
k
k
p
j
j
p
j
k
j
j
j
p
k
k
k
k
k
j
j
j
j
j

k
j
k
k
k
k
j
1n0–2n0
9n5–16n0
62–64
k
j
QMHE*
j
j
j
j
j
p
k
1n0
6
62–64
d
j
PLIE
k
k
k
j
k
k
j
1n0
6
61–65
d
j
OKHE
k


k

k

0n2–1n0
16n4–39n6
60–64
j


* Four letter codes were given to each cluster to represent the restriction patterns produced upon
digestion of the amplified 16S rDNA with MspI, HinfI, HhaI and Sau3AI, respectively. For each
enzyme, the different letters represent the different restriction patterns produced (not shown).
bium (de Lajudie et al., 1998a), Agrobacterium and
Rhizobium (Jordan, 1984)] (Fig. 2). Strain CCBAU
10071T, representing Cluster 2, was closely related to
B. japonicum and B. liaoningense.
respectively). DNA–DNA homology values between
the representative strains of Cluster 2 and the reference
strains of B. japonicum and B. elkanii ranged from 10
to 47n8 %.
DNA GjC content and DNA–DNA relatedness
determinations
RFLP analysis of 16S rDNA
Results of these analyses are shown in Table 2. The
DNA GjC contents of the Bradyrhizobium strains
ranged from 61n8 to 64n5 mol %, which is within the
range for recognized members of the genus Bradyrhizobium. The DNA–DNA homology values seen
among strains within each of the four clusters were
higher than 70 %, indicating that Clusters 1–4 represented four distinct genomic species (Graham et al.,
1991 ; Wayne et al., 1987). The mean DNA–DNA
homology values were 92n3, 83n2, 83n1 and 86n9 %
among the strains within Clusters 1, 2, 3 and 4,
respectively. Clusters 1, 3 and 4 were identified as S.
saheli, B. japonicum and B. elkanii, respectively, due to
the DNA homology values seen between the representative strains of the clusters and the type strains of
the aforementioned species (89n6, 100 and 91n1 %,
2226
The RFLP patterns produced for the 16S rDNA from
the Cluster 2 strains CCBAU 10011, CCBAU 10031,
CCBAU 10038, CCBAU 10040, CCBAU 10071T and
CCBAU 10073 were identical. Digestion of the 16S
rDNA of B. japonicum, B. elkanii and Cluster 2 strains
with Sau3AI produced identical patterns. Upon digestion of the 16S rDNA from strains of Clusters 1–4,
HhaI, MspI and HinfI produced distinctive patterns
for the strains of the different clusters. The 16S rDNA
from Cluster 2 strains and B. japonicum USDA 6T
produced the same patterns when digested with
Sau3AI or HhaI, but 1–2 bands differed between them
in the MspI- or HinfI-digested PCR products. Four
letter codes were given to each of the clusters, which
represented the patterns produced upon digestion of
the 16S rDNA with each of the four different endonucleases (Table 3 ; patterns not shown). For each
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Bradyrhizobium yuanmingense sp. nov.
enzyme, the different letters refer to the different
patterns produced.
Determination of cellular plasmid content
Six strains from Cluster 2 were checked ; no plasmids
were observed from strains CCBAU 10011, CCBAU
10031, CCBAU 10038, CCBAU 10040, CCBAU
10071T and CCBAU 10073.
Cross-inoculation tests
Strain CCBAU 10071T, representing Cluster 2, nodulated V. unguiculata and Glycyrrhiza uralensis, but it
did not nodulate Glycine max, Phaseolus vulgaris,
Pisum sativum, Galega officinalis, T. repens or Leucaena
leucocephala. S. saheli USDA 4102T, B. japonicum
USDA 6T and B. elkanii USDA 76T nodulated
Lespedeza cuneata. Two other strains isolated from
Lespedeza spp., USDA 3204 (Cluster 4) and USDA
3654 (Cluster 3), did not nodulate Glycine max.
DISCUSSION
The diversity and classification of rhizobial species
associated with Lespedeza spp. have not been investigated systematically. In this study, we characterized
rhizobial strains isolated from Lespedeza spp. using a
polyphasic approach, which included numerical taxonomic analysis, SDS-PAGE analysis of soluble proteins, DNA–DNA hybridization analysis, 16S rRNA
gene sequence analyses and nodulation tests with
selected host legumes. The results obtained from the
different methods used were in general agreement, and
four clusters were recognized among the strains on the
basis of these results. Besides the four clusters, some
single strain or small groups of (two to three) strains
were also identified. Some of the strains studied were
distinctive from all the type or reference strains used
here for recognized species, while others had more
than 80 % phenotypic similarity, as determined by
numerical taxonomical analysis, to B. japonicum, S.
saheli, S. terangae, S. fredii, R. galegae or Mesorhizobium tianshanense. These results indicate that Lespedeza spp. are associated with a number of different
species belonging to the genera Bradyrhizobium, Rhizobium, Sinorhizobium and Mesorhizobium ; however,
the single strain branches and small groups of strains
were not classified in this study. Therefore, Lespedeza
spp. are non-selective hosts for the microsymbionts
described here.
Bradyrhizobium strains were the predominant microsymbionts, nodulating Lespedeza spp. from the USA
soils (Table 1 ; Fig. 1). Most of the American strains
grouped within Clusters 3 and 4 and were identified as
B. japonicum and B. elkanii, respectively, on the basis
of the results from the different analyses performed.
Only four of the American Bradyrhizobium strains did
not belong to these two clusters. The Lespedezanodulating strains B. japonicum USDA 3204 and B.
elkanii USDA 3399 did not nodulate soybean, but the
soybean-derived strains B. japonicum USDA 6T and B.
elkanii USDA 76T formed nodules on L. cuneata.
These results may imply the existence of biovars within
Bradyrhizobium spp., but further data regarding the
host ranges of the different strains are needed before
biovars can be described. It has been reported that the
host ranges of Bradyrhizobium spp. are rather complicated. Strains isolated from a wide range of hosts in
China (Chen et al., 1991 ; Gao et al., 1994) have been
classified as B. japonicum.
Fast-growing, acid-producing rhizobial species were
isolated mainly from Chinese soils associated with nine
Lespedeza spp. (Table 1 ; Fig. 1). Most of these strains
grouped in Cluster 1 (S. saheli). S. saheli was originally
described for strains nodulating Sesbania spp. in Africa
(de Lajudie et al., 1994). The identification of Cluster 1
strains as Sinorhizobium saheli indicated a wide distribution of this bacterium, and added to it new hosts
native to the temperate regions of China. The nodulation observed upon inoculation of L. cuneata with
the type strain of S. saheli demonstrated that the
strains isolated from Sesbania spp. and the strains
isolated from Lespedeza spp. represent a cross-nodulating group of bacteria. It is interesting that species of
Sesbania and Lespedeza can share their microsymbionts, since these two groups of plants have geographical origins and habitats that are very different
from each other. In this case, it appears that the
symbiotic determinants of the plants may not relate to
the phylogeny of the legume species.
Based on our results, geographical restrictions exist for
the different rhizobial species that nodulate Lespedeza
spp. Although L. bicolor and L. daurica are found in
the USA and China, they nodulate with fast-growing,
acid-producing rhizobia in Chinese soils and with
Bradyrhizobium spp. in American soils. The differences
between the USA strains and the Chinese strains might
indicate a geographical isolation for these rhizobial
populations. In other words, there may be a lack of
natural transmission of these rhizobial species between
China and the USA. Strains associated with L.
inschanica were from China and were identified mainly
as Sinorhizobium saheli, but two strains grouped with
S. terangae and S. fredii (Fig. 1). L. juncea was found
to nodulate with both B. elkanii and B. japonicum in
the American soils. Although only strains of Bradyrhizobium spp. were isolated from L. juncea and L.
cuneata and strains of Sinorhizobium spp. were the
dominant organisms isolated from L. inschanica,
complex relationships should be expected between the
Lespedeza spp. and rhizobial species when more
rhizobial strains are investigated.
Diverse Bradyrhizobium populations and many novel
groups or lineages belonging to this genus have been
reported, including strains isolated from peanut (van
Rossum et al., 1995), shrubby legumes (Lafay &
Burdon, 1998), Acacia albida (Dupuy et al., 1994),
Aeschynomene spp. (van Berkum et al., 1995 ; Ladha &
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Z. Y. Yao and others
So, 1994 ; So et al., 1994) and Lupinus spp. (Barrera et
al., 1997). These reports indicated that novel taxa
should be described for these slow-growing, alkaliproducing rhizobia. In this study, Bradyrhizobium
strains isolated from L. cuneata growing in Beijing
formed a unique group (Cluster 2) based upon the
results of numerical taxonomic, SDS-PAGE, 16S
rDNA RFLP and DNA–DNA hybridization analyses.
The high 16S rRNA gene sequence similarity between
strain CCBAU 10071T (the representative strain of
Cluster 2) and B. japonicum LMG 6138T (Fig. 2)
indicated that Cluster 2 represented a lineage within
the genus Bradyrhizobium.
We have noticed that the criterion of 70 % DNA–DNA
homology as the cut-off value for strains of the same
species has been heavily criticized (Ward, 1998) ;
strains showing DNA–DNA homology values of much
less than 70 % have been included in some single
species, e.g. R. tropici type A and type B (Martı! nezRomero et al., 1991), Mesorhizobium plurifarium (de
Lajudie et al., 1998b) and R. mongolense (van Berkum
et al., 1998). These cases offer examples where the
definition of a species was not based solely on
DNA–DNA homology data, but also on phenotypic
distinctiveness and results from other genetic analyses.
In this study, strains within Clusters 1, 2, 3 and 4 had
DNA–DNA homology values of more than 72n2, 72n6,
71n4 and 72n0 %, respectively. Although DNA–DNA
homology values of up to 47n8 % were obtained among
the different clusters, there was a big gap between
the homologies within a genomic group and among
different groups. Therefore, it is clear from our
results that Cluster 2 represents a distinct genomic
species that is different from B. japonicum, B. liaoningense and B. elkanii. Since the strains within Cluster 2
could be distinguished from the three recognized
members of the genus Bradyrhizobium on the basis of
phenotypic features, SDS-PAGE analysis of proteins
and RFLP analysis of 16S rDNA, we propose that the
strains within this cluster represent a novel species of
the genus Bradyrhizobium, Bradyrhizobium yuanmingense. Features useful in distinguishing this novel
species from other Bradyrhizobium spp. can be found
in Table 3 ; another unique feature of this novel species
is that it can form ineffective nodules on Medicago
sativa and Melilotus albus, but not on soybean.
Optimum growth pH is between 6n5 and 7n5 ; no growth
at pH 5n0 or 10n0. Sensitive to 1n0 % (w\v) NaCl in
YMA. Reduces nitrate. Uses mannitol, pyruvate and
xylose, but not -ribose, sorbose, starch, -arginine,
glycine or -methionine as sole carbon source. Nitrate,
-alanine, -glutamine, -isoleucine, -threonine and
hypoxanthine are utilized as sole nitrogen source.
Sensitive to treatment at 60 mC. Catalase-positive and
-phenylalaninase-negative. Does not produce 3-ketolactose. Symbiotic genes are chromosomal-borne ; no
plasmids observed. Other characteristics of the species
can be found in Table 3. Isolated from L. cuneata, but
also nodulates V. unguiculata and Glycyrrhiza uralensis. DNA GjC content is 61n8–64n1 mol % (Tm).
The type strain of Bradyrhizobium yuanmingense is
CCBAU 10071T (l CFNEB 101T) ; generation time of
the type strain is 10n2 h and its DNA GjC content is
63n0 mol % (Tm).
ACKNOWLEDGEMENTS
This research formed a part of project no. 39130010,
supported by the National Science Foundation of China.
We thank Dr Peter van Berkum for supplying us with the
USDA isolates.
NOTE ADDED IN PROOF
While this manuscript was in preparation for publication,
Young et al. (2001) proposed a revision of the genus
Rhizobium, so that it would include all species of the genus
Agrobacterium and Allorhizobium undicola. Fig. 2 has been
changed to reflect the amended classification, as has the text
of the manuscript. The almost-complete 16S rRNA gene
sequence of B. liaoningense was also deposited within
GenBank during this time (Willems et al., 2001).
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